This application generally relates to genetic engineering and specifically involves the use of engineered animal cells containing an expressible target gene coupled to an expressible reporter gene to assay for mutagens.
Toxicity testing is required for many new drugs and agents released into the environment or work place. Compounds must generally be screened for damage to the embryo (teratogenesis activity) and for damage to the differentiated animal (carcinogenesis or mutagenesis activity).
Classically, compounds have been assayed for mutagenic activity using short term tests employing bacterial cell systems (STT) or animal studies. Most animal studies are conducted using the protocol for rodents developed by the National Cancer Institute in the early 1970s, reported by Sontag, et al., in U.S. Dep. Health Educ. Welfare Publ. (NIH) Carcinog. Tech. Rep. Serv. 1,76 (1976). However, the correlation between the results obtained in the two systems is poor and hard to measure objectively. For example, as reported by Tennant, et al., in Science 236, 933-941 (1987), there is only approximately 60% concordance between four widely used STTs and rodent carcinogenicity results. The four tests that were compared were the Salmonella mutagenesis, SAL, (described in Haworth, et al., Environ. Mutagen. 5 suppl. 1)3 (1983) and Mortelmans, et al. Toxicol. Appl. Pharmacol. 75, 137 (1984), etc.; chromosome aberrations in Chinese hamster ovary cells, ABS; sister chromatid exchanges in Chinese hamster ovary cells, SCE, (both described in Galloway, et al. Environ. Mutagen. 7, 1 (1985), and Galloway, et al. Environ. Mutagen (1987); and mouse lymphoma cell, MOLY, (described in Myhr, et al., Evaluation of Short-Term Tests for Carcinogens: Report of the International Programme on Chemical Safety's Collaborative Study on in vitro Assays, vol. 5 of Progress in Mutation Research Series, pp. 55-568, J. Ashby, et al. Editors (Elsevier, Amsterdam, 1985)) assays.
A recent variation of the SAL, or Ames, in vitro assay was reported by Oda, et al., in Mutation Research 147, 219-229 (1985). Oda, et al., introduced a fused umuC'-'lacZ gene into Salmonella typhimurium. The umu operon in Escherichia coli is responsible for chemical and radiation mutagenesis and is inducible by DNA-damaging agents. Induction of umu by the compound being tested is determined by production of beta-galactosidase activity resulting from expression of lacZ. A similar modification to the SOS chromotest to detect DNA-damaging agents, described by Quillardet, et al., Proc. Natl. Acad. Sci. (USA) 79, 5971-5975 (1982), uses one of the SOS genes, sfiA, fused to lacZ on the chromosome of E. coli.
There are disadvantages associated with the rodent assays which are distinct from those identified with the in vitro assays. The most significant problem is the length of time required to demonstrate that a compound is carcinogenic, since the determination is made based on tumor growth following exposure to the compound being tested. In general, animal studies must extend over a period of at least 12 to 18 months before a compound can be determined not to be carcinogenic. Another problem is that it is impossible to distinguish between genotoxic compounds (those inducing mutations within the DNA which leads to tumor growth) and those which alter some other non-DNA factor which leads to tumor growth. Still another problem with the rodent assay is that it is qualitative, not quantitative, making it possible only to determine the minimum dosage which induces tumor growth in the species being tested under the assay conditions.
To date, very few in vitro assays screen compounds for non-genotoxic carcinogenic activity. One in vitro assay is described by Penman and Fey in U.S. Pat. No. 4,569,916. This method is based on induction of morphological changes in a cell line upon exposure to a compound to be tested.
Compounds have traditionally been screened for teratogenesis activity, or inhibition of cell differentiation, by exposing an embryo to the compound to be tested, then examining samples from different areas of development for aberrations or inhibition of growth. Detailed descriptions of systems in use are reported by Flint in Teratology of the Limbs edited by Merker, et al., p. 325 (Walter de Gruyter & Co., Berlin 1980) and Flint, et al., J. Cell Sci. 61, 247 (1983); Concepts in Toxicology, Vol. 3 In Vitro Embryotoxicity and Teratogenicity Tests edited by Homburger, et al., (Karger, Basel 1985); Toxic. Appl. Pharmac. 76, 383 and J. Appl. Toxicol. 4, 109 (1984). As reported, assays for cytotoxicity and for cell differentiation are made in vitro, with confirming studies in vivo. When in vitro studies are conducted, at a minimum, cells from the midbrain and forelimb areas must be exposed to the compound and examined for detrimental effects. Another in vitro teratogenicity assay is reported by Keller in Molecular Toxicology 1, 261-276 (1987) using vaccinia virus growth in primate cell cultures, stated to be equally predictive of human teratogenesis as the in vivo rodent assay.
None of the methods presently in use for screening for carcinogenesis or teratogenesis activity in vitro correlate very well with in vivo results, or, where results can be correlated, the tests are time consuming, cannot be quantitated easily and are limited in sensitivity.
It is therefore an object of the present invention to provide a simple, quantifiable, more sensitive assay for mutagens, carcinogens, and teratogens.
It is another object of the present invention to provide an accurate measure of mutagenesis or teratogenesis activity that can be accurately correlated with in vivo observations.